For many years, horticulturist and agronomist have applied chemicals for weed control, plant protection and plant growth regulation by spraying the fields. For compositions that need to be applied on the plant, e.g., on the foliage, only a small part of the composition is bound to and retained on the part of the plant where it can exert its biological activity as large amounts are not adhering to the plant surface and are lost by drip-off or washed away by rain. Apart from giving rise to reduced efficacy of the chemical, losses of chemicals into the soil due to dripping off the plant while spraying or due to wash-out during rainfall may result in groundwater contamination, environmental damage, loss of biodiversity, and human and animal health consequences.
Several researchers have tried to solve this problem by applying slow release particles to the plant that stick to the leaves and release their content over a certain period of time. U.S. Pat. No. 6,180,141 describes composite gel microparticles that can be used to deliver plant-protection active principles. WO 2005102045 describes compositions comprising at least one phytoactive compound and an encapsulating adjuvant, wherein the adjuvant comprises a fungal cell or a fragment thereof. U.S. 20070280981 describes carrier granules, coated with a lipophilic tackifier on the surface, wherein the carrier granule adheres to the surface of plants, grasses and weeds.
Those microparticles, intended for the delivery of agrochemicals, are characterized by the fact that they stick to the plant by rather weak, aspecific interactions, such as a lipophilic interaction. Although this may have advantages compared with the normal spraying, the efficacy of such delivery method is limited, and the particles may be non-optimally distributed over the leaf, or washed away under naturally variable climatological conditions, before the release of the compound is completed. For a specific distribution and efficient retention of the microparticles, a specific, strongly binding molecule is needed that can assure that the carrier sticks to the plant till its content is completely delivered.
Cellulose binding domains (CBDs) have been described as useful agents for attachment of molecular species to cellulose (U.S. Pat. Nos. 5,738,984 and 6,124,117). Indeed, as cotton is made up of 90% cellulose, CBDs have proved useful for delivery of so called “benefit agents” onto cotton fabrics, as is disclosed in WO9800500 where direct fusions between a CBD and an enzyme were used utilizing the affinity of the CBD to bind to cotton fabric. The use of similar multifunctional fusion proteins for delivery of encapsulated benefit agents was claimed in WO03031477, wherein the multifunctional fusion proteins consist of a first binding domain which is a carbohydrate binding domain and a second binding domain, wherein either the first binding domain or the second binding domain can bind to a microparticle. WO03031477 is exemplified using a bifunctional fusion protein consisting of a CBD and an anti-RR6 antibody fragment binding to a microparticle, which complex is deposited onto cotton treads or cut grass. However, the use of such multifunctional fusion proteins for delivery of encapsulated benefit agents suffers from a number of serious drawbacks. First, although cellulose is a major component of plant cell walls and about 33% of all plant matter consists of cellulose, cellulose is, in intact living plants, shielded off from the outside environment by the plant cuticle, formed by cutin and waxes, which is an impermeable barrier with which plant cell walls are covered, making cellulose poorly accessible for binding by CBDs. Secondly, effective delivery of an encapsulated benefit agent to the plant requires simultaneous binding of the first binding domain to the plant and the second binding domain to the microparticle. As the likelihood of both binding events occurring is determined by a delicate equilibrium between the molar concentrations of the binding domains and their target molecules and the molar concentration of the bound complex, it is highly unlikely that sufficient multifunctional fusion proteins are present in solution to enable such simultaneous binding. Moreover, the equilibrium of a binding event is strongly influenced by environmental parameters such as temperature and pH, for which the optimal conditions may be considerably different for each of the binding domains. Therefore, it is highly unlikely that such simultaneous binding of two binding domains of such multifunctional fusion protein would result in a sufficiently strong binding that would retain an encapsulated benefit agent to a plant. Thirdly, although binding of a CBD is to a certain extent specific for cellulose, using a multifunctional fusion protein in which CBD should bind to the plant is to be considered as a generic binding approach, as all plants contain cellulose, and is therefore similar to aspecific sticking with tackifiers or stickers. A targeted approach in which specific binding of a binding domain would allow discrimination between binding to one plant species versus another would be of considerably higher value. WO03031477 also suggests, without further exemplification, that other binders to carbohydrates or polysaccharides can be used to generate fusion proteins to deposit microparticles onto living organisms. However, neither binding domains other than CBDs, nor binding domains binding to intact living plants were disclosed in WO03031477.
Molecules that are well known for their specificity and high affinity to particular targets are antibodies. Antibodies can be generated against a broad variety of targets, and antibodies that were generated to study plant cell wall architecture and dynamics have been described to bind specifically to particular plant constituents, predominantly constituents of the plant cell wall (Penell et al., 1989; Jones et al., 1997; Willats et al., 1998; Willats and Knox, 1999; Willats et al., 2001). However, it is unclear whether any of the plant cell wall constituents to which the antibodies have been generated, would be directly accessible for an antibody from the outside environment. Moreover, antibodies are by their very nature as components of the adaptive immune system construed such that they bind their targets under physiological conditions, including tightly regulated pH, temperature, and blood's normal osmolarity range. Should one consider to use antibodies for targeted delivery of agrochemicals, the antibodies should not only be capable of binding their target on an intact living plant in an agrochemical formulation, for which physicochemical characteristics deviate substantially from physiological conditions, they should also be capable to bind strongly enough to retain a carrier onto a plant. For neither of the plant-binding antibodies earlier described, either of these two crucial characteristics have been demonstrated.
The variable domains of camelid heavy chain antibodies (VHH) are a particularly interesting type of antibody fragments, as they are small, 15 kDa single-chain proteins, which can be selected for displaying high affinity for their targets. Also, by their nature as small single-chain molecules, VHH are easy to produce and have superior stability characteristics over conventional antibodies. However, so far, no plant-binding VHH have been described. Moreover, although VHH that are covalently linked to a solid resin particle have been shown to maintain functionality in the sense that they are able to capture antigen from a solution (WO 0144301), it has not been shown, nor can it be expected, that the affinity of VHH for its target is sufficient to retain a carrier onto a solid plant surface.